A recombinant porcine circovirus type 3 trimer protein and its preparation method and application

Abstract

The present invention discloses a recombinant porcine circovirus type 3 trimer protein, a preparation method and an application thereof. The present invention uses bioinformatics methods and resources to predict the B cell antigen epitopes and T cell antigen epitopes of the PCV3Cap protein. Taking into account the stability and immunogenicity of the antigen epitopes, a recombinant porcine circovirus type 3 trimer protein based on the PCV3Cap protein antigen epitope is designed, and its efficient soluble expression in Escherichia coli is achieved. The expressed recombinant porcine circovirus type 3 trimer protein can be mass-produced and purified by Ni-NTA affinity chromatography. The purified recombinant porcine circovirus type 3 trimer protein can assemble into a stable trimer structure. The subunit vaccine prepared using the recombinant porcine circovirus type 3 trimer protein can induce experimental pigs to produce a high level of antibodies and has a significant protective effect on the experimental pigs. The recombinant porcine circovirus type 3 trimer protein designed by the present invention provides a new idea for the development of PCV3 vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering vaccines, and in particular relates to a recombinant porcine circovirus type 3 trimer protein and a preparation method and application thereof. Background Art

[0002] Porcine circovirus (PCV), belonging to the genus Circovirus in the family Circoviridae, is considered one of the most economically devastating viral pathogens worldwide. PCV1 is considered nonpathogenic to pigs, while PCV2 is the primary cause of multisystemic wasting syndrome (PMWS) in piglets. PCV3 was first reported in the United States in 2016 and has since been detected on farms in several countries, including Poland, South Korea, Italy, Brazil, and China. PCV3 has been associated with porcine dermatitis and nephropathy syndrome, congenital tremor, reproductive disorders, and multisystem inflammatory diseases. Although PCV3 and PCV2 have been found to share similar syndromes, the capsid protein sequence of PCV3 shares low homology with PCV2. PCV2 vaccines do not provide cross-protection against PCV3 infection, and PCV3 antisera do not cross-react with PCV1 or PCV2 samples. Therefore, further molecular and structural understanding of the pathogenesis of PCV3 is needed to inform the design and development of PCV3 diagnostic kits and vaccines.

[0003] Porcine circovirus type 3 (PCV3) is a non-enveloped, single-stranded circular DNA virus, one of the smallest DNA viruses. Its genome contains two major open reading frames (ORFs). ORF1 encodes replication-related proteins, and ORF2 encodes the capsid protein. The PCV3 Cap protein can self-assemble into virus-like particles (VLPs) with T=1 icosahedral symmetry, similar to PCV2. The assembly of PCV3 VLPs may provide clues for the design and development of effective PCV3 vaccines. Researchers have expressed the PCV3 Cap protein in Escherichia coli, yeast, and insect expression systems. However, the PCV3 Cap protein expressed in these systems either forms inactive inclusion bodies or is expressed at very low levels, detectable only by Western blotting. Achieving a sufficient yield of pure PCV3 Cap requires complex purification processes, and the high cost makes it difficult to mass-produce and promote animal vaccines. Due to the difficulty in isolating and culturing PCV3 strains and the difficulty in large-scale production of PCV3 Cap protein expressed in various expression systems, no PCV3 vaccine is currently available. Summary of the Invention

[0004] Currently, PCV3 strains are difficult to isolate and culture for inactivated vaccine production, and PCV3 Cap proteins expressed in various expression systems have low yields, poor stability, and high production costs, making large-scale production of subunit vaccines difficult. To address the current lack of PCV3 vaccines, the present invention utilizes bioinformatics methods and resources to predict B-cell and T-cell epitopes of the PCV3 Cap protein. Taking into account the stability and immunogenicity of the epitopes, a recombinant porcine circovirus type 3 trimer protein based on the PCV3 Cap protein epitope was designed and efficiently expressed in Escherichia coli. The expressed recombinant porcine circovirus type 3 trimer protein can be mass-produced and purified by Ni-NTA affinity chromatography. The purified recombinant porcine circovirus type 3 trimer protein can assemble into a stable trimer structure. The subunit vaccine prepared using this recombinant porcine circovirus type 3 trimer protein can induce high antibody levels in experimental pigs and has a significant protective effect on the experimental pigs. The recombinant porcine circovirus type 3 trimer protein designed in the present invention provides a new approach to the development of PCV3 vaccines.

[0005] One of the objectives of the present invention is to provide a recombinant porcine circovirus type 3 trimer protein, the amino acid sequence of which is shown in Sequence 1, comprising: a PCV3 polypeptide epitope, a trimer domain, and a sequence connecting the PCV3 polypeptide epitope and the trimer domain: GTAGGGSG; the trimer domain is a chicken cartilage matrix protein, the amino acid sequence of which is shown in Sequence 8;

[0006] PCV3 polypeptide epitopes include four antigenic epitope polypeptides: Epitope1, Epitope2, Epitope3, and Epitope4, whose amino acid sequences are shown in Sequence 3, Sequence 4, Sequence 5, and Sequence 6, respectively;

[0007] and a universal T cell antigen epitope polypeptide Epitope5, whose amino acid sequence is shown in SEQ ID NO: 7, for activating CD4+ cells;

[0008] Epitope1, Epitope2, Epitope3, Epitope4, and Epitope5 are connected by the amino acid sequence GGGGSGGGGS.

[0009] The second object of the present invention is to provide a nucleotide sequence for expressing the recombinant porcine circovirus type 3 trimer protein, as shown in Sequence 2.

[0010] The method for preparing the recombinant porcine circovirus type 3 trimer protein provided by the present invention comprises the following steps:

[0011] Step 1. PCR amplification was performed using the nucleotide sequence shown in SEQ ID NO: 2 as a template to obtain the PCV3-Trimer gene fragment;

[0012] Step 2. Ligate the PCV3-Trimer gene fragment and the linearized pET28a vector, transform the fragment into Escherichia coli DH5a competent cells, culture a single colony, perform colony PCR identification, and screen positive clones to obtain the recombinant expression plasmid pET28a-PCV3-Trimer.

[0013] Step 3. The recombinant expression plasmid pET28a-PCV3-Trimer was transformed into Escherichia coli Rosetta (DE3) to obtain the expression strain pET28a-PCV3-Trimer. The expression strain was inoculated into liquid LB medium containing kanamycin and cultured on a shaker at 37°C to obtain a seed solution.

[0014] Step 4: Inoculate the seed solution into the fermentation tank at a ratio of 3% and culture until the OD 600 When the value is 20, add IPTG with a final concentration of 0.5 mM, and ferment for 8 to 10 hours at pH 7.0, dissolved oxygen not less than 30%, and 25°C;

[0015] Step 5. The fermentation broth is centrifuged to collect the bacteria, and the lysis solution is added to resuspend the bacteria, and the high-pressure homogenization is performed to break the bacteria. The supernatant is collected by centrifugation, and the impurities and endotoxins are removed by Ni-NTA affinity chromatography to obtain the purified recombinant porcine circovirus type 3 trimeric protein.

[0016] Furthermore, the step of removing impurities and endotoxins from the lysis supernatant in step 5 includes: clarifying the supernatant through a 0.45 μm hollow fiber column to reduce turbidity and then loading it onto chromatography, washing the chromatography column with an imidazole washing solution containing 0.1% Triton X114 to wash away impurities and endotoxins; washing the chromatography column with a lysis buffer to wash away residual Triton X114; and eluting the recombinant porcine circovirus type 3 trimer protein with an imidazole eluent, collecting the eluate for desalting and filtration sterilization.

[0017] The third object of the present invention is to provide the use of the recombinant porcine circovirus type 3 trimer protein in subunit vaccines.

[0018] 1. The present invention predicts the B cell epitopes and T cell epitopes of the PCV3 Cap protein through bioinformatics methods, and designs a recombinant porcine circovirus type 3 trimeric subunit vaccine. The antigen used in this vaccine is in the form of a trimer, which has better immunogenicity and stability.

[0019] 2. The recombinant porcine circovirus type 3 trimer antigen designed by the present invention can be efficiently expressed in Escherichia coli in a soluble manner, and the expression level in high-density fermentation can reach 2 g / L;

[0020] 3. The recombinant porcine circovirus type 3 trimer antigen designed by the present invention can be prepared on a large scale by simple Ni-NTA affinity chromatography, and the endotoxin content is less than 25 EU / ml;

[0021] 4. The porcine circovirus type 3 trimeric subunit vaccine designed by the present invention has a significant protective effect on experimental pigs, and the vaccine is prepared by an Escherichia coli expression system, with a simple production process and low cost, meeting the production needs of animal vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Design of expression construct for recombinant porcine circovirus type 3 trimeric subunit vaccine;

[0023] Figure 2 Agarose electrophoresis diagram of the results of restriction enzyme digestion of pET28a vector and PCR amplification of PCV3 trimer gene; lane 1 is the linearized pET28a vector after restriction enzyme digestion; lane 2 is the linearized PCV3 trimer fragment obtained by PCR amplification; lane M is the DL5000 marker; Figure 3 Agarose electrophoresis diagram showing the PCR identification results of PCV3 Trimer monoclonal recombinants: Lanes 1, 2, 3, 4, and 5 are the PCR results of different monoclonal colonies; Lane 6 is the PCR negative control; Lane 7 is the PCR positive control; Lane M is the DL2000 marker;

[0024] Figure 4 The results of SDS-PAGE detection of the recombinant expression plasmid pET28a-PCV3-Trimer transformed into Escherichia coli Rosetta (DE3) for induction expression; lanes 1, 2, and 3 are the total protein, supernatant protein, and precipitated protein after induction, respectively; lane M is a protein marker; Figure 5 Western Blot analysis results of expression strains pET28a, pET28a-PCV3-Trimer, and pET28a-PCV3 induced expression; lanes 1, 2, and 3 represent the total protein after induced expression of expression strains pET28a, pET28a-PCV3-Trimer, and pET28a-PCV3, respectively; lane M is a protein marker;

[0025] Figure 6 The results of SDS-PAGE detection of PCV3-Trimer shake flask-induced expression and purification; lane 1 is the supernatant protein of bacterial lysis; lane 2 is the purification flow-through; lane 3 is the purification eluate; lane M is a protein marker;

[0026] Figure 7 For the analysis and identification of PCV3-Trimer; Figure 7-A. Activity analysis of PCV3-Trimer by agarose gel electrophoresis; Figure 7 -B. Western Blot analysis of PCV3-Trimer; lane 1: PCV3-Trimer treated with reducing agent DTT; lane 2: purified and assembled PCV3-Trimer; lane M: protein marker;

[0027] Figure 8 The figure shows the results of PCV3-Trimer high-density fermentation SDS-PAGE detection; lane 1 is a 250 μg / ml BSA standard; lane 2 is a 125 μg / ml BSA standard; lane 3 is the total protein of the fermentation cell lysate; lane 4 is the precipitated protein of the fermentation cell lysate; lane 3 is the supernatant protein of the fermentation cell lysate; lanes 6, 7, 8, and 9 are 2-fold serial dilution samples of the supernatant protein of the fermentation cell lysate; lane M is a protein marker;

[0028] Figure 9 The results of SDS-PAGE and activity agarose gel electrophoresis for PCV3-Trimer purified by high-density fermentation were shown. Figure 9 -A. SDS-PAGE detection of 2-fold serial dilution of PCV3-Trimer; Figure 9 -B. Detection of purified PCV3-Trimer by activity agarose gel electrophoresis. Lane 1 shows purified assembled PCV3-Trimer; lane 2 shows PCV3-Trimer treated with 5 mM DTT; lane 3 shows PCV3-Trimer treated with 1 mM DTT; lane M shows a protein marker.

[0029] Figure 10 The indirect ELISA method was used to detect the antibody levels in the serum of experimental pigs;

[0030] Figure 11 Agarose gel electrophoresis diagram of PCR detection of PCV3 in pig sera before and after challenge. Lanes 1, 2, 3, and 4 represent pre-challenge serum samples; lanes 5, 6, 7, and 8 represent post-challenge serum samples; lane 9 represents a negative control; lane 10 represents a positive control; lane M represents a DL2000 marker.

[0031] Figure 12 To detect PCV3 antigen in the lymphoid tissue of experimental pigs by immunohistochemistry. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited thereto.

[0033] The main materials used in the examples of the present invention are as follows: Escherichia coli Rosetta (DE3) (Merck), pET28a plasmid (Merck), DNA polymerase, endonuclease, T5 exonuclease, etc. were purchased from New England Biolabs, and primers were purchased from Shanghai Sangon Biotechnology Co., Ltd.; dithiothreitol (DTT) was purchased from Thermo Fisher Scientific; lysis buffer: 20 mM Tris-HCl, pH 8.0; imidazole wash solution: 60 mM imidazole, 0.1% Triton X114, 20 mM Tris-HCl, pH 8.0; imidazole elution solution (500 mM imidazole, 20 mM Tris-HCl, pH 8.0), 1 M IPTG solution. The expression strains pET28a and pET28a-PCV3-Cap were previously constructed and preserved.

[0034] Example 1 Design of recombinant porcine circovirus type 3 trimeric subunit vaccine

[0035] Based on the amino acid sequence of PCV3 Cap protein published by NCBI (GenBank: QKZ25703.1), the ABCpred and BepiPred programs were used to analyze the continuous and discontinuous B-cell epitopes of the PCV3 Cap protein. As shown in Table 1, 10 high-scoring, surface-exposed B-cell epitopes were pre-selected. Furthermore, the IEDB program was used to predict seven high-scoring T-cell epitopes, most of which were located within B-cell epitopes. For example, the T1 epitope is located within the B3 epitope, the T2 epitope is located within the B6 epitope, the T3 epitope is located within the B5 epitope, the T4 epitope is located within the B7 epitope, the T5 epitope is located within the B4 and B7 epitopes, and the T6 epitope is located within the B2 epitope.

[0036] Table 1 Prediction of PCV3 Cap T cell antigen epitopes and B cell antigen epitopes

[0037] Serial number B cell antigen epitope amino acid sequence Serial number T cell antigen epitope amino acid sequence B1 NNKPWHANHFITRLNE T1 ETAISFEYY B2 TWLQDDPYAESSTRKV T2 TSKKKHSRY B3 SFEYYKILKMKVTLSP T3 TVQWGALLW B4 SRPTPWLNTYDPTVQW T4 SAHPGQSLF B5 LNTYDPTVQWGALLWS T5 RPTPWLNTY B6 HSRYFTPKPILAGTTS T6 NTWLQDDPY B7 GQSLFFFSRPTPWLNT T7 AIDLDGAWT B8 AGTYYTKKYSTMNVIS B9 YVPEKTGMTDFYGTKE B10 ALLWSIYVPEKTGMTD

[0038] 2. To induce both humoral and cellular immune responses, e.g. Figure 1 , designed 5 peptides containing 8 linear B cell antigen epitopes and 7 T cell antigen epitopes; one of them also includes a universal T cell antigen epitope (PADRE) that activates CD4+ cells to enhance helper T cell activity.

[0039] Table 2 Design of expression structure of recombinant porcine circovirus type 3 trimeric subunit vaccine

[0040]

[0041] As shown in Table 2 and Figure 1 Epitope1 contains the B8 antigen epitope in Table 1; Epitope2 contains the B1, B3, and T1 antigen epitopes in Table 1; Epitope3 contains the B2, B6, T2, T6, and T7 antigen epitopes in Table 1 in its amino acid sequence; Epitope4 contains the B4, B5, B7, T3, T4, and T5 antigen epitopes in Table 1; Epitope5 contains the universal T cell antigen epitope (PADRE) that activates CD4+ cells; the trimer domain (Trimer) is chicken cartilage matrix protein (CMP), which can form a stable trimer structure; the expression structure of the recombinant porcine circovirus type 3 trimer subunit vaccine is designed as shown in the following figure. Figure 1 As shown, PCV3 polypeptide epitopes are linked together by using (GGGGS)2, and the PCV3 polypeptide epitope portion is linked to the chicken cartilage matrix protein trimer base sequence (CMP) by GTAGGGSG; in addition, 6xHis is added to the C-terminus of the trimer sequence for purification of the recombinant porcine circovirus type 3 trimer protein (PCV3-Trimer).

[0042] The amino acid sequence of the PCV3-Trimer designed in the present invention is shown in SEQ ID NO: 1. This sequence enables the recombinant PCV3-Trimer protein to assemble into a stable trimer structure, thereby improving the stability of the recombinant protein. In addition, multimeric antigens have better immunogenicity than oligomeric antigens and can induce higher antibody levels.

[0043] Table 3 Positions of each polypeptide in sequence 1

[0044]

[0045] Example 2 Construction of PCV3-Trimer Prokaryotic Expression Vector

[0046] According to the PCV3-Trimer amino acid sequence designed in Example 1, the PCV3-Trimer gene was synthesized by codon optimization in Escherichia coli, and PCR amplification was performed using primers F1 and R1 to obtain the PCV3-Trimer gene fragment, as shown in Sequence 2. Figure 2 Lane 2, primer design as follows (underlined portion is homologous to the vector):

[0047] F1:5'- AGGAGATATACCATG GCTGGCACCTATTACACCAAAAAGT-3',R1:5'- GGTGGTGGTGCT CGA GTTAGTGATGGTGATGGTGATGGCC-3',

[0048] The extracted pET28a plasmid was double-digested with NcoI and XhoI and recovered. The digestion results are shown in Figure 2 Lane 1; PCV3-Trimer gene fragment obtained by PCR amplification and pET28a vector linearized by enzyme digestion were reacted on ice with T5 exonuclease at a molar ratio of 3:1 for 5 min. The product was transformed into Escherichia coli DH5a competent cells by conventional methods, and single colonies were picked for colony PCR identification ( Figure 3 ), screening positive clones, Figure 3 Lanes 1, 2, 3, 4, and 5 are positive clones identified by PCR. The extracted plasmids were sent to the company for sequencing, and the recombinant expression plasmid pET28a-PCV3-Trimer was obtained.

[0049] Example 3 Expression and Identification of PCV3-Trimer

[0050] The recombinant expression plasmid pET28a-PCV3-Trimer constructed in Example 2 was transformed into Escherichia coli Rosetta (DE3) to obtain the recombinant expression strain pET28a-PCV3-Trimer. The expression strain was inoculated into liquid LB medium containing kanamycin and cultured on a shaker at 37°C and 220 rpm until the OD 600 When the value was 0.6, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 25 ° C and 220 rpm for 8 h. The bacteria were collected by centrifugation and re-selected with lysis buffer. The bacteria were crushed by high-pressure homogenization and the supernatant and precipitate were separated by centrifugation. The expression of PCV3-Trimer was detected by SDS-PAGE. The results are shown in Figure 4 A target band (~30kDa) consistent with the expected size of PCV3-Trimer appeared, and most of the PCV3-Trimer appeared in the bacterial lysis supernatant in a soluble form. The results showed that PCV3-Trimer can be efficiently expressed in Escherichia coli, and the soluble expression amount is about 35% of the total soluble protein.

[0051] The above expression strain pET28a-PCV3-Trimer and the previously constructed and preserved strains pET28a and pET28a-PCV3-Cap were induced to express, respectively. The induced proteins were extracted and subjected to SDS-PAGE, and then transferred to a PVDF membrane. PCV3-positive pig serum was used as the primary antibody and HRP-labeled goat anti-pig IgG was used as the secondary antibody. The expressed PCV3-Trimer was identified by Western Blot. The results are shown in Figure 2. Figure 5As shown, the expression strains pET28a-PCV3-Trimer and pET28a-PCV3-Cap both produced obvious target bands, while no specific bands were detected in the strain pET28a, indicating that the expressed PCV3-Trimer (~30kDa) and PCV3 Cap (~25kDa) can be recognized by PCV3-positive pig serum and are both immunoreactive.

[0052] Example 4 Purification of PCV3-Trimer and Identification of PCV3-Trimer Trimer Assembly

[0053] The expression strain pET28a-PCV3-Trimer was inoculated into 400 ml of liquid LB medium containing kanamycin and cultured on a shaker at 37°C and 220 rpm until the OD 600 When the value was 0.6, IPTG was added to a final concentration of 0.5 mM, and the culture was continued for 8 hours at 25°C in a shaker at 220 rpm. The cells were collected by centrifugation, resuspended in 40 ml of lysis buffer, broken by high-pressure homogenization, and the supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane. 5 ml of Ni-NTA filler was loaded into the chromatography column, and the column was rinsed with 5 column volumes of pure water to remove ethanol at a flow rate of 1.5 ml / min. The column was equilibrated with 5 column volumes of lysis buffer at a flow rate of 1.5 ml / min. The filtered supernatant protein was passed through the column at a flow rate of 1 ml / min. The column was washed with 5 column volumes of imidazole washing solution containing 0.1% Triton X114 to remove impurities and endotoxins at a flow rate of 2 ml / min. Residual Triton X114 was removed with 5 column volumes of lysis buffer at a flow rate of 2 ml / min. The PCV3-Trimer protein was eluted with 3 column volumes of imidazole elution solution at a flow rate of 2 ml / min. The purification results are shown in FIG. Figure 6 As shown, through one-step purification by Ni-NTA, the purity of PCV3-Trimer protein can reach more than 90%, the concentration is 1.2 mg / ml, and the endotoxin content is less than 50 EU / ml.

[0054] The purified PCV3-Trimer protein was divided into two tubes, treated with the strong reducing agent 1mM DTT (dithiothreitol) and left untreated. The PCV3-Trimer protein in each tube was then analyzed by agarose gel electrophoresis and Western Blot. The conditions for agarose gel electrophoresis were: 100mM histidine, 100mM MES, pH 6.1; 1% agarose; electrophoresis at 100V for 90 minutes. The results of agarose gel electrophoresis and Western Blot were as follows: Figure 7As shown, the PCV3-Trimer protein in the group treated with the strong reducing agent DTT was in monomeric form, while the PCV3-Trimer without DTT treatment showed obvious aggregates, indicating that the purified PCV3-Trimer protein can assemble into stable trimers.

[0055] Example 5 Large-scale fermentation and purification of PCV3-Trimer

[0056] The expression strain pET28a-PCV3-Trimer obtained in Example 3 was inoculated into a large shake flask, and the seed solution obtained was inoculated into a 500L fermenter at a ratio of 3%. The initial culture temperature was 37°C. After 5-6 hours of culture, the OD 600 When the value is 20, IPTG with a final concentration of 0.5mM is added, and the temperature is lowered to 25°C and induced for 8 to 10 hours. During the entire fermentation process, the pH is controlled at about 7.0 using ammonia water through an automatic acid-base adjustment device, and the dissolved oxygen is controlled at more than 30% by controlling the rotation speed and the ventilation device. The carbon source and nitrogen source are supplemented according to the changes in pH and dissolved oxygen. After the fermentation is completed, the bacteria are collected by centrifugation using a disc centrifuge, resuspended with 500L lysis buffer, crushed by high-pressure homogenization, and sampled for centrifugation to separate the supernatant and precipitated protein. The fermentation expression of PCV3-Trimer protein is detected by SDS-PAGE. The results are as follows Figure 8 As shown, the PCV3-Trimer protein expression strain can efficiently and solublely express PCV3-Trimer protein during fermentation culture, with the expression level reaching 2 g / l.

[0057] The fermentation lysate supernatant was clarified through a 0.45 μm hollow fiber column to reduce turbidity and then loaded onto a chromatographic column at a flow rate of 60 L / h. The column was washed with 3 to 5 column volumes of imidazole wash solution containing 0.1% Triton X114 until the UV value stabilized at a flow rate of 200 L / h to remove contaminants and endotoxins. The column was washed with 3 to 5 column volumes of lysis buffer until the UV value stabilized at a flow rate of 200 L / h to remove residual Triton X114. The PCV3-Trimer protein was eluted with imidazole eluent at a flow rate of 60 L / h, and the elution peak was collected. The eluate was desalted, sterilized by 0.22 μm filtration, and stored at 4°C.

[0058] The purified protein sample was taken and its endotoxin content was determined by limulus amebocyte lysate reagent, and the purified PCV3-Trimer was detected by SDS-PAGE and agarose gel electrophoresis respectively; the SDS-PAGE results were as follows: Figure 9 A, PCV3-Trimer protein purity reached 95%, concentration was 1mg / ml, and endotoxin content was less than 25EU / ml; agarose gel electrophoresis results were as follows Figure 9B, The purified PCV3-Trimer protein is in the form of a trimer and can form oligomers under treatment with a strong reducing agent. The results are consistent with those of laboratory purification, indicating that the purified PCV3-Trimer can assemble into stable trimers and can be produced on a large scale industrially.

[0059] Example 6 Preparation of recombinant porcine circovirus type 3 trimeric subunit vaccine and animal immunization

[0060] The purified antigen PCV3-Trimer protein was diluted to 0.4 mg / ml, and water adjuvant was added according to the ratio of antigen solution: vaccine adjuvant = 4:1 (w / w) to prepare the finished vaccine for subsequent immune challenge experiments, wherein the antigen content of the finished vaccine was 80 μg / ml. Serum was collected from 3-week-old experimental pigs, and anti-PCV3 antibodies were determined by ELISA kits, and PCV3 nucleic acid was determined by PCR. Twelve experimental pigs with negative antibodies and nucleic acids were selected and randomly divided into 3 groups: 4 blank control groups, which were not treated; 4 unrelated control groups, which were injected with 2 ml of normal saline into the neck muscle; 4 experimental groups, which were injected with 2 ml of recombinant porcine circovirus type 3 trimer subunit vaccine into the neck muscle. Serum samples of all experimental pigs were collected 14 days and 21 days after immunization, respectively, and the antibody levels of the experimental pigs were detected by ELISA kits. The results are shown in the table. Figure 10 The experimental group of piglets produced high levels of antibodies 14 days after immunization, and the antibody titer reached more than 1:5000 on 21 days, while the PCV3 antibodies in the control group were all negative. The results showed that the subunit vaccine made of recombinant porcine circovirus type 3 trimeric protein can induce the experimental pigs to produce high levels of antibodies.

[0061] Example 7 Challenge protection experiment of recombinant porcine circovirus type 3 trimeric subunit vaccine

[0062] 3.0 g of inguinal lymph node tissue samples from pigs that were positive for PCV3 antibodies and nucleic acid and had no contamination by other pathogens were collected. The samples were rinsed in ice-cold saline to remove blood, dried with filter paper, and added with 20.0 ml of sterile saline. The samples were ground with a tissue grinder at 10,000-15,000 r / min to prepare a tissue homogenate. The homogenate supernatant was collected after centrifugation at 10,000 r / min for 5 min and filtered through a 0.22 μm filter for sterilization. 100 μL of the homogenate supernatant was used to extract viral DNA using a viral nucleic acid extraction kit. The PCV3 copy number was detected by qPCR using primers F2: 5'-CTACGAGTGTCCTGAA-3'; R2: 5'-CCTCCACACTCCACAATA-3'. The PCV3 copy number was 5×10 8 The filtered homogenate supernatant was used to perform a challenge experiment on the pigs in the irrelevant control group and the immune experimental group in Example 6, with 1 ml injected intravenously to each pig, and 1 ml of sterile PBS was injected intravenously to each pig in the blank control group.

[0063] After 21 days of challenge, serum was collected from all experimental pigs, and the pigs were sacrificed and dissected for tissue samples. Nucleic acid was extracted from the serum of all experimental pigs before and after challenge, and PCV3 nucleic acid was detected by PCR. The primers used were F3: 5'-GCTACGAGTGTCCTGAAGATAAG-3'; R2: 5'-GCCTCCACAC TCCACAATAG-3'; PCR test results are shown in Figure 2. Figure 11 No PCV3-specific bands were detected in the experimental pigs in the vaccine-immunized challenge group and the blank control group, while PCV3 was detected in all the non-immunized challenge groups, indicating that the serum viral load of the experimental pigs in the vaccine-immunized challenge group was significantly reduced, and the porcine circovirus type 3 trimeric subunit vaccine had a significant protective effect on the experimental pigs.

[0064] Lymphatic tissue sections were taken from all experimental pigs and PCV3 antigens were detected in lymph nodes by immunohistochemistry. Figure 12 No obvious PCV3 antigen was detected in the group immunized with the recombinant porcine circovirus type 3 trimer subprotein vaccine and the blank control group, while obvious PCV3 antigen was detected in the non-immunized group, indicating that the porcine circovirus type 3 trimer subunit vaccine has a significant protective effect on the experimental pigs. This result is consistent with the viremia test results.

Claims

1. A recombinant porcine circovirus type 3 trimeric protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 1, and includes: a PCV3 polypeptide epitope, a trimer domain, and a sequence connecting the PCV3 polypeptide epitope and the trimer domain: GTAGGGSG; the trimer domain is a chicken cartilage matrix protein, and its amino acid sequence is shown in SEQ ID NO: 8; PCV3 polypeptide epitopes include four antigenic epitope polypeptides: Epitope1, Epitope2, Epitope3, and Epitope4, whose amino acid sequences are shown in Sequence 3, Sequence 4, Sequence 5, and Sequence 6, respectively; and a universal T cell antigen epitope polypeptide Epitope5, whose amino acid sequence is shown in SEQ ID NO: 7, for activating CD4+ cells; Epitope1, Epitope2, Epitope3, Epitope4, and Epitope5 are connected by the amino acid sequence GGGGSGGGGS.

2. A nucleic acid molecule for expressing the recombinant porcine circovirus type 3 trimer protein according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in Sequence 2, and the nucleic acid molecule is used to prepare a recombinant porcine circovirus type 3 trimer protein.

3. The method for preparing the recombinant porcine circovirus type 3 trimer protein according to claim 1, wherein The following steps are involved: Step 1. PCR amplification was performed using the nucleotide sequence shown in SEQ ID NO: 2 as a template to obtain the PCV3-Trimer gene fragment; Step 2. Ligate the PCV3-Trimer gene fragment and the linearized pET28a vector, transform the fragment into Escherichia coli DH5a competent cells, culture a single colony, perform colony PCR identification, and screen positive clones to obtain the recombinant expression plasmid pET28a-PCV3-Trimer. Step 3. The recombinant expression plasmid pET28a-PCV3-Trimer was transformed into Escherichia coli Rosetta (DE3) to obtain the recombinant expression strain pET28a-PCV3-Trimer. The expression strain was inoculated into liquid LB medium containing kanamycin and cultured on a shaker at 37°C to obtain a seed solution. Step 4. Inoculate the seed solution at a ratio of 3% into a fermenter for cultivation. When the OD600 value reaches 20, add IPTG to a final concentration of 0.5 mM. Ferment and cultivate at pH 7.0, dissolved oxygen not less than 30%, and 25°C for 8-10 hours. Step 5. The fermentation broth is centrifuged to collect the bacteria, and the lysis solution is added to resuspend the bacteria, and the high-pressure homogenization is performed to break the bacteria. The supernatant is collected by centrifugation, and the impurities and endotoxins are removed by Ni-NTA affinity chromatography to obtain the purified recombinant porcine circovirus type 3 trimeric protein.

4. The method according to claim 3, characterized in that The step of crushing the supernatant to remove foreign proteins and endotoxins in step 5 includes: clarifying the supernatant through a 0.45 μm hollow fiber column to reduce turbidity and then loading it on chromatography, washing the chromatography column with an imidazole washing solution containing 0.1% Triton X114 to wash away foreign proteins and endotoxins; washing the chromatography column with a lysis buffer to wash away residual Triton X114; and eluting the recombinant porcine circovirus type 3 trimer protein with an imidazole eluent, collecting the eluate for desalting and filtration sterilization.

5. Use of the recombinant porcine circovirus type 3 trimer protein according to claim 1 in the preparation of a subunit vaccine for preventing PCV3 infection.

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